The invention relates to the use of peptides, proteins, and other oligomers to provide a means by which normally quenched nanoparticle fluorescence may be recovered upon detection of a target molecule. Further, the invention provides a structure and method to carry out detection of target molecules without the need to label the target molecules before detection.
Nanotechnology research and its findings have been studied in many different research areas and are being applied for the development of scientific and industrial technologies, such as nano electronics, sensors, and catalysts. Frontier energy, medical, and security-related nanotechnology will depend on the integration and optimization of nanoparticle-based technologies and biological sciences to design hybrid materials for increasingly cleaner and more efficient energy conversion and storage, as well as biological sensors having increased sensitivity. Such integration requires not only the precise control of nanoparticle size, shape, composition, and surface properties, but also the ability to self-assemble and dissociate nanoparticles with controlled kinetics and final assembly morphology under conditions tolerated by biological systems.
Currently, the self-assembly of nanoparticles with metallic (Au, Ag, Pt), semiconductive (CdSe, CdS, ZnS, GaAs), and magnetic (Fe2O3) properties using biological building blocks is achieved by two main approaches: (i) DNA-based systems and (ii) protein- (peptide-) based systems. The remarkable specificity and programmable interactions of DNA allows self-assembly of DNA-conjugated nanoparticles and construction of complex architectures. Their complexity and functionality may be extended via the incorporation of proteins that function as biological sensors or as organizers of complex scaffolds. The DNA-based self-assembly systems have shown great potential as biological sensors. Specific binding of peptides to inorganic surfaces has been demonstrated, and these peptides can be selected by using phage display system. Also, the peptides have been successfully used for the construction of nanostructures and self-assembly of inorganic nanoparticles. Due to the functional variety of proteins, the combination of DNA- and protein-based systems is receiving considerable attention to design functional hybrid nanomaterials.
The self-assembly of nanoparticles and programmed complex architectures using DNA have been demonstrated. DNA-induced self-assembly of nanoparticles was first introduced in 1996 by pioneering papers of Mirkin and co-workers, “A DNA-based method for rationally assembling nanoparticles into macroscopic materials,” Nature, 382(6592): p. 607-609, 1996, and Alivisatos and co-workers, “Organization of ‘nanocrystal molecules’ using DNA,” Nature, 382(6592): p. 609-611, 1996, both of which articles are hereby incorporated by reference in their entirety. The ability of specific hybridization of DNA was utilized for self-assembly of nanoparticles on which single stranded DNA (ssDNA) is chemically immobilized. Aggregation was obtained by adding a single stranded linker DNA whose ends were complementary to the ssDNAs conjugated to the particles. The aggregation of ssDNA conjugated nanoparticles is accompanied by changes of physical and optical properties, and was applied to detect DNA. Addition of a DNA fragment complementary to the linker results in dissociation of the aggregates. Alternatively, the system can be designed such that the DNA fragments to be detected function as the linker. The melting properties of DNA, which depend on sequence and length, make it possible to reverse the self-assembly of the aggregates by increasing temperatures (FIG. 1). The sequence-dependent melting properties allow detection of single point mutation using ssDNA-conjugated nanoparticles.
Gold-nanoparticle quenched fluorescent oligonucleotides, that are designed for complementary binding at their 3′ and 5′ ends to form a hairpin structure, have been used as molecular beacons for detecting target DNA that hybridizes to the hairpin structure, resulting in emission of the quenched fluorescence.
Microarray technology is used on a routine basis for high through-put quantification of large numbers of different DNA or RNA fragments. The array-based systems require labeling of target DNA, or RNA, via synthesis of C-DNA. ssDNA-conjugated nanoparticles have been used as candidates to probe their target molecules.
In addition, the specific interaction of DNA has been used to create DNA building blocks, and assembly of the building blocks to construct sophisticated geometries and morphologies.